Novel in-vitro models and correlative experiments with primary tumor/normal tissue specimens have been utilized to identify microRNA (miR) alterations which contribute to initiation and progression of tobacco -associated thoracic malignancies. These experiments identified several micro-RNAs that are either up-regulated or silenced in normal respiratory epithelia and lung cancer cells by cigarette smoke. For example, cigarette smoke condensate (CSC) mediated epigenetic repression of miR-487b in normal respiratory epithelia and lung cancer cells. Basal levels of miR-487b were significantly lower in lung cancer cells relative to normal respiratory epithelia. qRT-PCR and RNA-CLIP experiments demonstrated that miR-487b directly targets transcripts encoding Wnt5a, polycomb group proteins SUZ12 and BMI1, as well as c-Myc and K-ras proto-oncogenes in SAEC and lung cancer cells. All of these targets have been implicated in the establishment of a stem-cell phenotype in pulmonary carcinomas. Expression levels of miR-487b were significantly lower in resected lung cancers (especially those from smokers) relative to adjacent normal lung tissues. Repression of mir-487b correlated significantly with over-expression of all five targets in primary lung cancers. Constitutive expression of miR-487b significantly decreased expression of the five targets, and decreased proliferation, tumorigenicity and in-vivo invasion/metastasis of lung cancer cells. Collectively these experiments were the first to demonstrate that mir-487b is a novel tumor suppressor silenced by epigenetic mechanisms during human pulmonary carcinogenesis. Results of these experiments were recently published in the Journal of Clinical Investigation. In additional experiments, array and qRT-PCR techniques were used to examine miR expression in immortalized esophageal epithelia (IEE) and esophageal adenocarcinoma (EAC) cells cultured in normal media (NM) with or without CSC. Under relevant exposure conditions, CSC significantly decreased miR-217 expression in these cells. Endogenous levels of miR-217 expression in EAC cells (EACC)/ primary EACs were significantly lower than those observed in IEE/ paired normal esophageal tissues. Subsequent experiments demonstrated direct interaction of miR-217 with kallikrein 7 (KLK7), encoding a putative oncogene not previously implicated in EAC. Repression of miR-217 correlated with increased levels of KLK7 in primary EACs, particularly those from smokers. Additional experiments demonstrated that CSC-mediated repression of miR-217 coincided with DNMT3b-dependent hypermethylation and decreased occupancy of nuclear factor 1 (NF-1) within the miR-217 genomic locus. Deoxyazacytidine induced miR-217 expression, and down-regulated KLK7 in EACC; deoxyazacytidine also attenuated CSC-mediated miR-217 repression and up-regulation of KLK7 in IEE and EACC. Over-expression of miR-217 significantly decreased, whereas over-expression of KLK7 increased proliferation, invasion and tumorigenicity of EACC. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of EAC via up-regulation of KLK7, and suggest that restoration of miR-217 expression may be a novel treatment strategy for these malignancies. A manuscript summarizing these experiments has been submitted for publication. In additional experiments, we sought to examine the frequency and potential clinical relevance of telomerase complex mutations in esophageal carcinomas after identifying a unique germ line telomerase RNA component (TERC) deletion in a patient undergoing surgery for Barrett's adenocarcinoma. Briefly, sequencing techniques were used to evaluate mutational status of telomerase reverse transcriptase (TERT) and TERC in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. These experiments identified one deletion involving TERC (TERC del 341-360), and two non-synonymous TERT variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p 0.01). Subsequent experiments demonstrated that relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-beta-catenin complex, markedly depleted beta-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. Fluorescence in-situ hybridization and spectral karyotyping experiments demonstrated that A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage. Collectively, these experiments were the first to identify a telomerase variant in a human malignancy that simultaneously disrupts canonical as well as non-canonical telomerase activities. Our findings support further analysis of variant/mutations involving telomerase complex in esophageal cancers and preneoplastic esophageal lesions. Results of these studies were recently published in PLoS One. Our published experiments have demonstrated that aberrant expression of Polycomb Repressive-Complex-2 (PRC-2)- a mediator of stem cell pluripotency, contributes to initiation and progression of lung cancers via epigenetic repression of tumor suppressor genes such as Dkk1. Furthermore, we have demonstrated that one strategy to efficiently inhibit PRC-2 activity in thoracic malignancies involves pharmacologic depletion of EZH2, a core component of PRC-2. Another potential strategy involves targeting PRC-2 associated proteins, such as JARID2, which recruits PRC-2 to DNA. Recent qRT-PCR and immunoblot experiments have demonstrated significant over-expression of JARID2 and EZH2 in cultured lung cancer cells/primary lung cancers relative to normal respiratory epithelial cells/tissues. Biochemical knockdown of JARID2 did not significantly alter EZH2 or other PRC-2 components; similarly knockdown of EZH2 did not affect JARID2 expression in lung cancer cells. Knockdown of JARID2 or EZH2 inhibited proliferation of lung cancer cells by 70-80%, and significantly decreased growth of xenografts relative to vector controls. Under exposure conditions achievable in clinical settings, mithramycin dramatically depleted JARID2 in a dose-and time-dependent manner, and recapitulated the in-vitro and in-vivo inhibitory effects of JARID2 or EZH2 knockdown. A variety of tumor suppressor stem cell polycomb target genes were up-regulated in lung cancer cells/xenografts following JARID2 or EZH2 knockdown or mithramycin treatment. Collectively, these experiments were the first to demonstrate that JARID2 is over-expressed in lung cancers, and that pharmacologic depletion of JARID2 is a novel strategy to target PRC-2 for lung cancer therapy. A manuscript pertaining to these experiments is being prepared for publication.